WO2020096004A1 - Animal cell growth promoter, culture medium for animal cell culture, and animal cell culture apparatus - Google Patents

Abstract

[Problem] The purpose of the present invention is to provide a novel animal cell growth promoter. Specifically, a culture supernatant of an embryonic membrane of a bird or reptile egg is used as an animal cell growth promoter. For example, the growth of animal cells can be promoted by the addition, to a culture medium for animal cell culture, of a cell growth agent that contains as an active ingredient the culture supernatant of an embryonic membrane of a bird or reptile egg.

Description

Animal cell growth promoter, animal cell culture medium and animal cell culture device

The present invention relates to an animal cell growth promoter, an animal cell culture medium, and an animal cell culture device.

In culturing animal cells, a serum medium in which fetal bovine serum or the like is added to the medium as a factor having a function of maintaining cell growth or a serum-free medium in which hormones or growth factors are added in place of serum is generally used. ..

In addition to these factors, Japanese Patent Laid-Open No. 2013-247927 discloses that rice bran extract has a growth promoting action on cultured animal cells. Further, Japanese Patent Laid-Open No. 2011-182736 discloses that the cell growth rate can be increased by culturing animal cells in a medium containing a hydrolyzate of a β-conglycinin concentrate. Further, JP-A-7-188292 discloses that a glycoprotein contained in the culture supernatant of human-derived fibroblasts promotes the growth of human vascular endothelial cells and hepatocytes. Further, JP-A-5-301893 discloses that a protein contained in a cell culture supernatant obtained by culturing a human-derived glioma cell line has a growth promoting effect on glial cells and fibroblasts.

The object of the present invention is to provide a novel animal cell growth promoter, animal cell culture medium and animal cell culture device.

One embodiment according to the present invention is an animal cell growth promoter containing, as an active ingredient, the culture supernatant of the embryonic membrane of an avian or reptile egg. The egg may be a chicken egg. The animal cell growth promoter may be a cell growth agent for muscle cells, visceral cells, or nervous cells, or a cell growth agent for liver cells, pancreatic cells, or oviduct cells.

Another embodiment of the present invention is a cell culture medium containing the cell growth promoter described in any of the above.

Another embodiment according to the present invention is a first culture tank for culturing cells derived from embryonic membrane of an avian or reptile egg, a second culture tank for culturing animal cells for the purpose of proliferation, and A first flow path for flowing the medium from the first culture tank to the second culture tank; a second flow path for flowing the medium from the second culture tank to the first culture tank; The cell culture medium is refluxed in the order of the first flow channel, the second culture tank, and the second flow channel, and the first flow channel and the second flow channel are set according to the state of the animal cells and / or the cell culture medium. An animal cell culture device comprising: a medium flow rate control unit that controls the flow of the cell culture medium in a flow path. In addition, a medium introduction path for introducing a fresh cell culture medium into the refluxing cell culture medium, and a medium removal for removing the cell culture medium after culturing from the refluxing cell culture medium The medium flow rate control unit further comprises a passage, and the medium flow control unit introduces the fresh cell culture medium from the medium introduction passage and the medium removal passage according to the state of the animal cell and / or the cell culture medium. The removal of the culture medium for cell culture after the culture may be controlled.

== Cross reference with related documents ==
This application claims priority based on Japanese Patent Application 2018-210910 filed on Nov. 8, 2018, and is incorporated herein by reference to the basic application.

It is a schematic diagram of the cell proliferation apparatus in one embodiment of the present invention. In one embodiment of the present invention, when the culture supernatant of chicken chorioallantoic membrane is added to the medium, the tissue shown in the figure (stomach, skeletal muscle, heart, gallbladder, intestine, brain, bursa of Fabricius and bone) is derived. 5 is a photograph of observation results showing that cell proliferation is promoted in cells of No. 1 is a graph quantitatively showing the results of FIG. 1 for liver, pancreas, and oviduct-derived cells in one example of the present invention. In one embodiment of the present invention, cells derived from the tissues shown in the figure (stomach, muscle, heart, liver, intestine, brain, Fabricius bursa and bone), when the culture supernatant of chicken egg yolk sac is added to the medium 3 is a graph showing that cell proliferation is promoted by.

The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention, and are provided for the purpose of illustration or explanation, and the present invention is not limited thereto. It is not limited. It will be apparent to those skilled in the art that various alterations and modifications can be made based on the description of the present specification within the intention and scope of the present invention disclosed in the present specification.

== Animal cell growth promoter ==
The animal cell growth promoter of the present disclosure contains a culture supernatant of an embryonic membrane of an egg of a bird or a reptile as an active ingredient.

Birds or reptiles that are the origin of the eggs that collect the germinal membranes include, but are not limited to, quail, chickens, lizards, snakes, crocodiles, turtles, etc., and are birds or reptile animals that have germinal membranes of eggs. I wish I had it.

Here, the embryonic membrane refers to a membrane tissue that is formed outside the embryo body during the development process of birds and reptiles, and has functions such as protection of the embryo, nutrition and respiration, and is not involved in the construction of the body after hatching or birth. Meaning, for example, the serosa located on the outermost side of the embryo, the amniotic membrane that wraps the embryo body, the allantois that creates the urinary sac, the chorioallantoic membrane (CAM) in which the serosa and allantois are partially adhered, and the yolk. Examples include the yolk sac that wraps.

The culture supernatant of the germinal membrane is a medium in which the germinal membrane is isolated and cultured. The germinal membrane to be cultured may be one kind or a mixture of plural kinds among the above-mentioned membranes such as serosa, amniotic membrane, allantoic membrane, allantoic membrane and yolk sac.

The embryonic membrane can be collected from the egg by a known method (for example, see Eiji Ichishima: Chemistry and Biology, 13: 8, p489-497 (1975)). For example, specifically, the embryo membrane can be isolated by the following method. First, after fertilized eggs are incubated for a predetermined period under conditions suitable for artificial hatching, the eggshell is split, and (1) the allantoic membrane is obtained by taking out the swollen membrane generated from the ventral side of the posterior part of the digestive tract of the embryo. (2) The amniotic membrane can be obtained by taking out the transparent membranous tissue around the fetus in which blood vessels are not running, and (3) the yolk sac is the membranous tissue surrounding the yolk. (4) The serosa can be obtained by removing the outermost membranous tissue surrounding all the elements in the egg. Further, when the incubation time is extended, the serosa and the allanto partially fuse to form the chorioallantoic membrane. At this time, the chorioallantoic membrane is in contact with the eggshell and can be obtained by taking out the membrane tissue in which blood vessels are running. The optimal incubation period may be determined depending on each animal and the target membrane tissue. For example, in the case of chicken eggs, 4 days to 21 days are preferable, 10 days to 18 days are more preferable, and 13 days to 15 days is more preferred. When culturing these embryonic membranes, they may be cultured in a membranous state, but by physically disrupting the membrane or performing enzymatic treatment with a protease (eg, collagenase, elastase, dispase, papain). Alternatively, cells derived from the embryonic membrane may be isolated and cultured.

For culturing the embryonic membrane, a medium used for general animal cell culture can be used, and examples thereof include, but are not limited to, DMEM and F12. To this medium, supplements usually added as a medium additive may be added. Regarding the culture conditions, the conditions used for usual animal cell culture can be used, and typically, a carbonate buffer medium is used for culturing at 5% CO 2, 37 ° C. It is not limited and can be appropriately selected by those skilled in the art. The culture time is not particularly limited, but is preferably 1 to 7 days, more preferably 2 to 6 days. After culturing, the supernatant may be collected and added to the cell culture medium as it is, but a medium obtained by removing solid matters such as cell debris by filtration or centrifugation may be added. Good. The amount of the culture supernatant added to the cell culture medium is not particularly limited as long as it is an effective amount at which the growth effect of the cultured cells is recognized, but the final concentration in the medium is preferably 20% or more, more preferably 35% or more. , 50% or more is more preferable.

The type of animal cells to be cultured in the cell culture medium to which this culture supernatant is added is not particularly limited, and muscle cells such as smooth muscle cells, cardiomyocytes and skeletal muscle cells, heart cells, hepatocytes, cells derived from the stomach Examples thereof include visceral cells such as cells derived from intestines, and neural cells such as nerve cells and glial cells. The animal species from which the cells are derived is not particularly limited, and examples thereof include humans, mice, rats, monkeys and pigs.

In this way, by adding the culture supernatant obtained by culturing cells derived from embryonic membrane to the animal cell culture medium, it is possible to promote the growth of the cultivated animal cells. Therefore, this culture supernatant can be used as an animal cell growth promoter.

When using the culture supernatant of cells derived from embryonic membrane as an animal cell growth promoter, other cell growth factors may be used at the same time.

== Cell proliferation device ==
The cell expansion device of the present invention comprises a first culture tank for culturing cells derived from embryonic membrane of an avian or reptile egg, a second culture tank for culturing animal cells for the purpose of proliferation, and a first culture. A first channel for flowing the medium from the tank to the second culture tank, a second channel for flowing the medium from the second culture tank to the first culture tank, a first culture tank, and a first flow The cell culture medium is refluxed in the order of the channel, the second culture tank, and the second channel, and the first channel and the second channel are flown according to the state of the animal cells and / or the cell culture medium. And a medium flow rate control unit that controls the flow of the cell culture medium.

In the first culture tank, culture according to the method for culturing cells derived from embryonic membrane, which is performed when preparing the animal cell growth promoting agent as described above, is performed. In the second culture tank, culturing according to the culturing method of culturing animal cells in the medium to which the animal cell growth promoter is added as described above is performed.

The first culture tank and the second culture tank are fluidly connected by the first flow path and the second flow path. For example, as shown in FIG. 1A, the first culture tank 11 and the second culture tank 12 may be directly connected by the first flow channel 21 and the second flow channel 22. As long as the first culture tank 11 and the second culture tank 12 are fluidly connected, one or a plurality of other culture tanks may be provided, and the other culture tank and the first culture tank 11 or Various configurations can be considered for connection with the two culture tanks 12. For example, as shown in FIG. 1 (B), a third culture tank 13 is provided in the middle of the first flow path 21, and the flow path extends from the first culture tank 11 through the third culture tank 13 to the second culture tank 13. It may be connected to the tank 12. Alternatively, as shown in FIG. 1 (C), a third culture tank 13 is provided independently of the second culture tank 12, and the flow channels are the first flow channel 21 and the second flow channel 22. Separately, a third channel 31 and a fourth channel 32 that fluidly connect the first culture tank 11 and the third culture tank 13 may be provided. In the case of (B) and (C), by appropriately selecting the cells to be cultured in the third culture tank 13, the cells are cultured in the first culture tank 11 or the second culture tank 12. Can affect or influence their culture.

Here, the influence of the culture in the first culture tank 11 and the culture in the culture tanks other than the second culture tank 12 from the culture in the first culture tank and the culture in the second culture tank, There are various possible effects on the culture in the first culture tank and the culture in the second culture tank. For example, cell growth, cell differentiation, tissue morphogenesis, pH adjustment of the medium, Examples include prevention of bacterial contamination.

For example, specifically, by putting in the third culture tank 13 cells that are different from the cells in the second culture tank 12 and that can grow in a medium in which cells derived from germinal membrane are cultivated, The cells can be simultaneously grown in the second culture tank 12 and the third culture tank 13. Alternatively, by culturing cells derived from the embryonic membrane different from the first culturing tank 11 in the third culturing tank 13, it is possible to further promote the growth of the cells cultivated in the second culturing tank 12. it can. Alternatively, even if the cells cultured in the third culture tank 13 are cells capable of growing in a medium in which cells derived from germinal membranes are cultured and secreting factors that grow the cells cultured in the second culture tank 12. Good.

The tanks may be connected to each other by a pipe, and the pipe may form a flow path. A valve may be installed in these pipes. The medium flow rate control unit monitors the state of the cells being cultured and / or the medium for cell culture, and according to the state, the flow of the medium for cell culture in each flow path through this valve (for example, flow velocity, flow rate, etc.). ) May be controlled.

The structure of the valve is not particularly limited as long as it is a well-known structure such as a flange type or a screw type, and the method for adjusting the valve is not particularly limited, and a known structure such as an air-driven type, an electromagnetic type, an electric type, or a hydraulic type is used. Any adjustment method may be used. Further, in order to prevent the backflow of the cell culture medium in the tube, the tube may be provided with a check valve.

Examples of the state of the cells and / or cell culture medium monitored by the medium flow rate control unit include, but are not limited to, the state of cell proliferation, the state of cell differentiation, and the pH of the medium. The state of cell proliferation can be determined by, for example, counting the number of cells per unit area or measuring the ratio of the area occupied by cells to the bottom area of the plate. The state of cell differentiation can be determined by measuring fluorescence by, for example, introducing into the cells a marker gene that expresses upon differentiation and emits fluorescence. Regarding the pH of the medium, for example, the pH may be directly measured, or a pigment that changes depending on the pH may be added to the medium and the color may be used for the determination.

(1) Isolation method of chorioallantoic membrane or yolk sac The chorioallantoic membrane or yolk sac used in the test was isolated by the following method, respectively.

A fertilized egg of a chicken is incubated at 37 ° C, and 14 days later, the eggshell is broken, and the membrane tissue in contact with the eggshell and in which blood vessels are running is taken out as chorioallantoic membrane, while the membrane surrounding the yolk is taken out. The tissue was taken out as an yolk sac and each of these membrane tissues was immersed in HBSS (Hank's Balanced Salt Solution). These membrane tissues were physically finely cut with tweezers or the like, and then centrifuged to remove the supernatant.

A collagenase solution (200 IU / mL) was added to each isolated membrane tissue, and the mixture was kept at 37 ° C for 1 hour and then centrifuged again to remove the supernatant. HBSS was added to each collagenase-treated membrane tissue, filtered through a 40 μm filter, and the resulting filtrate was centrifuged to remove the supernatant to collect chorioallantoic membrane-derived cells or yolk sac-derived cells. When the chorioallantoic membrane-derived cells or yolk sac-derived cells are preserved, the cells are suspended in a cell freezing solution (10% DMSO solution) and frozen by gradually cooling to -80 ° C, and then -80 ° C. Saved in.

(2) Method for culturing chorioallantoic membrane or yolk sac The chorioallantoic membrane-derived cell or yolk sac-derived cell obtained in (1) contains 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) solution In addition to the prepared DMEM (hereinafter referred to as FBS / PS-containing DMEM), the cells were seeded in a cell culture dish (diameter 10 cm) at a concentration of 4 × 10 ⁶ cells / dish, and the amount was 2 to 3 at 37 ° C. and 5% CO ₂ environment. Cultured for 4 days.

(3) Method for recovering culture supernatant The chorioallantoic membrane culture solution or yolk sac culture solution prepared by the culture method shown in (2) was filtered using a 0.22 μm filter, and the obtained filtrate was used as an animal cell growth medium. Used as a promoter in animal cell culture. When the prepared culture supernatant was stored, it was frozen at -20 ° C and stored.

(4) Animal cell culture (4-1) Method for isolating cells from each tissue Chicken cells used for culture were isolated by the following method.

First, fertilized chicken eggs were incubated at 37 ° C, and 14 days later, the eggshells were broken and the chicken embryos were taken out. A stomach, skeletal muscle, heart, gall bladder, intestine, brain, bursa of Fabricius and bone were isolated from the embryonic day 14 chicken, and each was immersed in an HBSS solution. Then, cells derived from each tissue were isolated by the same method as in (1).

(4-2) Method for culturing cells from each tissue Cells derived from each tissue are cultured by adding an equal amount of the chorioallantoic membrane culture supernatant or the yolk sac culture supernatant prepared in (3) to FBS / PS-containing DMEM. Culture was performed in the same manner as in (2) using the culture medium. As a control, cells cultured under the same conditions were used, except that the chorioallantoic membrane culture supernatant or yolk sac culture supernatant was not added and the medium containing only FBS / PS-containing DMEM was used.

(4-3) Counting the number of cells in each culture The results of (4-2) were quantitatively analyzed for cells derived from liver, pancreas, oviduct, stomach, muscle, heart, intestine, brain, bursa of Fabricius and bone. Evaluated to. Here, the number of cells derived from each tissue was measured by the following method.

First, the culture solution of cells derived from each tissue was removed from the dish, and PBS was added to wash the cells. After removing the PBS, a trypsin-EDTA solution was added and the cells were detached by incubating at 37 ° C. for 1 to 2 minutes, and then FBS / PS-containing DMEM was added to stop the reaction. All the solution containing the detached cells was collected and centrifuged to obtain cells. Further, FBS.PS was added to the cells to suspend the cells, and 20 μL of the cell suspension was mixed with 20 μL of trypan blue solution. The number of live cells not stained with trypan blue in this cell suspension was counted under a microscope using a hemocytometer.

(4-4) Results First, cells were observed with a phase-contrast microscope when cultured in a medium supplemented with chorioallantoic membrane culture supernatant for about 3 to 5 days. Fig. 2 shows a photograph of the cells. In any cell culture, the cells with the chorioallantoic membrane culture supernatant prepared in (3) were added more than with the cells without the chorioallantoic membrane culture supernatant. There were many. This indicates that the growth of cells was promoted by the added chorioallantoic membrane culture supernatant.

In order to quantitatively show these results, FIG. 3 shows the average number of cells and the standard error in the case of using the chorioallantoic membrane culture supernatant for cells derived from the liver, pancreas, and oviduct in three petri dishes. However, in all cases of liver, pancreas, and oviduct-derived cells, the addition of chorioallantoic membrane culture supernatant resulted in approximately 2.5 to 4 times more cells than the case without addition of chorioallantoic membrane culture supernatant. Was growing.

Next, using a medium supplemented with the yolk sac culture supernatant, the growth promoting effect on cells of the stomach, muscle, heart, liver, intestine, brain, bursa of Fabricius and bone was also examined (n = 2). ), As shown in FIG. 4, in each case, the number of cells in the case of adding the yolk sac culture supernatant was higher than that in the case of not adding the chorioallantoic membrane culture supernatant. This indicates that cell culture was promoted by the added yolk sac culture supernatant.

The present invention has made it possible to provide a novel animal cell growth promoter, an animal cell culture medium, and an animal cell culture device.

Claims (8)

Animal cell culture growth promoter containing, as an active ingredient, the culture supernatant of embryonic membrane derived from fertilized eggs of birds or reptiles The animal cell proliferation promoter according to claim 1, wherein the embryonic membrane is at least one selected from the group consisting of amniotic membrane, chorioallantoic membrane and yolk sac. The animal cell growth promoter according to claim 1 or 2, wherein the egg is a chicken egg. The animal cell growth promoter according to any one of claims 1 to 3, which is a cell growth agent for muscle cells, visceral cells, or nervous cells. The animal cell growth promoter according to any one of claims 1 to 3, which is a cell growth agent for liver cells, pancreatic cells, and oviduct cells. A cell culture medium containing the cell growth promoter according to any one of claims 1 to 5. A first culture vessel for culturing cells derived from the embryonic membrane of a bird or reptile egg;
A second culture vessel for culturing animal cells for the purpose of proliferation,
A first flow path for flowing a medium from the first culture tank to the second culture tank;
A second flow path for flowing the medium from the second culture tank to the first culture tank;
The cell culture medium is refluxed in the order of the first culture tank, the first flow path, the second culture tank, and the second flow path, and the first culture tank is returned according to the state of the animal cells and / or the cell culture medium. And a medium flow rate control unit that controls the flow of the cell culture medium in the second flow channel and the second flow channel. A medium introduction path for introducing a fresh cell culture medium into the refluxing cell culture medium,
Further comprising a medium removal path for removing the cell culture medium after culturing from the cell culture medium to be refluxed,
The medium flow rate control unit introduces the fresh cell culture medium from the medium introduction path and cell culture after the culture from the medium removal path according to the state of the animal cells and / or the cell culture medium. The animal cell culture device according to claim 7, which controls the removal of the culture medium.

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